Tissues and organs have long been commonly transplanted for a number of indications. One of the well-established techniques is autotransplantation, where the patient's own tissue (eg. skin, bone, vein, or fat tissue) from one location is used to replace the tissue in another place. This is not always possible, however, and in a number of situations the patient needs to receive a transplant (eg. heart, kidney, retina, and others) from a suitable donor. The main problems of these so-called allotransplants are tissue rejection, and increasingly, lack of donors due to highly increasing demand. Therefore there is an effort to substitute natural allotransplants in various ways. For example, it is possible to culture autotransplants from the patient's cells using tissue engineering. These autotransplants easily overcome the immunity barrier, however they have certain disadvantages: the necessity to harvest tissue from the patient (biopsy), laborious and expensive cultivation and a long time lapse between biopsy and the transplant's application. This technique is commonly used when replacing skin in case of burns of 3rd degree, while in case of other tissues and organs, this technique is, at this point, experimental. For example, U.S. Pat. Nos. 6,878,383; 6,432,710; 5,858,390; 5,665,372 and 5,660,850 (Boss, Jr. et al.) describe techniques and means for the implantation of autologous fibroblasts in order to produce hyperplasia of patient's tissue.
Autotransplantation using artificially created epidermal skin layer has been used in burn patients for a number of years. In the year 1979, Rheinwald and Green (Green H, et al., Proc. Nat. Acad. Sci. USA, 1979; 76: 5665-8.) developed a method for the serial cultivation of human keratinocytes for autotransplantation. Since 1981, autologous cultivated epidermal grafts have been used in the USA for the healing of extensive burns (O'Connor N E, et al., Lancet 1981; 1: 75-8). The disadvantage of the method is the long time interval necessary for the cultivation of autologous keratinocytes, furthermore the fragility of the cultivate, difficult manipulation, high sensitivity to antibiotics, infection and other stresses and difficult evaluation of the assimilation of the graft (Navsaria H A, et al., Trends in Biotechnology 1995; 15: 91-100). Various improvements of the method are therefore described, such as:
U.S. Pat. No. 4,299,816 (M. G. Eisinger) describes modified healing of burns using grafts from artificially cultivated epidermal cells. U.S. Pat. No. 5,716,411 (Orgill et al) describes a healing method leading to skin regeneration for burns and injuries, using a biosynthetic cover consisting of a collagen matrix and glycoaminoglycanes, which allows the penetration of cells and blood vessels from the healing tissue on one side, and the application of sheets of autologous keratinocytes on the other side. WO 2006/107188 A1 (L. Lurvink et al.) describes a non-porous polypeptide film suitable for cell cultivation, and its subsequent use for the healing of wounds and burns. A recent overview of these methods can be found in TISSUE ENGINEERING Vol. 12, No. 9, 2006 Update on Tissue-Engineered Biological Dressings, M. Ehrenreich and Z. Ruszczak.
Not only autologous, but also allogenic cultivated epidermal grafts have a high healing effect in deep dermal burns, biopsy sites, crural ulcers and other skin defects (Bolivar-Flores J, et al., Burns 1990; 16: 3-8.; Matouskova E. et at., Burns 1993; 19: 118-23.4, 5).
The success of the procedure depends also on the selection of donor cells. P. Brychta et al. describe in the Czech Patent No. CZ 282711 a cultivated epidermal allotransplant from embryonic or fetal cells for the healing of skin defects and wounds, essentially according to the procedure of Reinwald and Green but while using allogenic cells, which are well received by the patient.
There is also an effort to increase mechanical resistance and viability of keratinocytes (eg. by cultivation on a synthetic substrate) and to develop techniques, which would enable permanent assimilation of the cultivated tissue onto 3rd degree burns. One example of a substrate used for the cultivation of keratinocytes is a membrane based on hyaluronic acid (Laser skin, FIBIA, Italy), various types of collagen matrixes combines with fibroblasts or also various substrates made of synthetic polymers (eg. experimental pHEMA at the Clinic of Burn Medicine, FNKV in Prague 10). To fill deep burns, dermal substitutes are developed such as Integra (collagen combined with glucose aminoglycan chondroitin-6-sulphate and allogenic fibroblasts; Integra LifeSciences Corporation, Plainsboro, N.J., USA), Dermagraft (polygalactin seeded with dermal allogenic fibroblasts; Advanced Tissue Sciences, La Jolla, Calif., USA), or the already mentioned AlloDerm—frozen allogenic dermis (LifeCell Corporation, The Woodlands, Tex., USA). However, all these dermal substitutes must be covered by a thin autologous dermo-epidermal graft during the second step (after 2-3 weeks of vascularization), the covering of 3rd degree burns using allogenic cultivated was not successful so far.
Another solution to the problems of allotransplantation is the use of tissue or organs from other species other than human, the so-called xenotransplantation. In this case it is also necessary to overcome the rejection of foreign tissue by the immune system, and it is also necessary to prevent the possibility of contamination of disease-causing microbes, germs, and viruses, from the donor to the patient. A great deal of attention is directed to prevent the possibility of transmission of prions from animals to man (eg. the famous “mad cow disease”). On the other hand, the advantage lies in the fact that animal tissues and organs are a lot more accessible than human ones.
A well-known example of xenotransplant is porcine heart valves, used to replace human heart valves. Porcine valves are cross-linked using glutaraldehyde (eg. U.S. Pat. No. 4,076,468, Liotta et al.; U.S. Pat. No. 4,247,292. W. A. Angell), which leads to several desirable results: the rejection reaction of the organism is suppressed, the hydrolytic and enzymatic stability of the xenotransplant is increased, and furthermore, glutaraldehyde acts as a chemical sterilizing agent. One of the disadvantages of this method is the change of mechanical properties of the tissue and in some cases even a long-term release of toxic glutaraldehyde from the insoluble polyaldehydes, which can form during the process and cannot be removed through simple extraction.
A significant portion of transplants is used in the form of so-called “biological covers” for wound dressing and the resultant healing support. Depending on the nature of the wound and other circumstances, biological, synthetic, and semi-synthetic covers are used. Biological covers are generally considered to be the most effective. A typical biological cover for the healing of, for example, burns, is mammalian skin, but especially human skin (allotransplant), or pig skin (xenotransplant) in various thicknesses, harvested from dead individuals and stored fresh under cold temperatures for a short period of time, or for even a longer period of time when frozen. Much experience was derived by xenotransplants from pig skin.
“Live” wound dressings (i.e. unprocessed allotransplants or xenotransplants containing all components of live skin) are very effective, but their disadvantage lies in their limited shelf life and in the possibility of transfer of infection. Certain solutions were suggested in a number of patents, eg. products AlloDerm and XenoDerm by the LifeCell Corp., Texas, USA, based on a cryopreservation method according to U.S. Pat. No. 4,865,871 (S. Livesey et al.). This method enables freezing and possibly freeze-drying tissues and cells without damaging their structure or function.
Another method is the storage of pig skin in glycerine in the presence of silver nitrate at room temperature, as described in the patent application CN 19951010722 (Kai Cao).
Sterilization of pig skin (after being cleaned and processed using hydrocarbons), in a sodium perchlorate or hydrogen peroxide solution using gamma radiation from a Co60 source is described in a patent application TW 199001117733 (Chang Hong Chi et al).
Other methods for the sterilization of pig skin for medical use are described in patent application CN 19921005926 (Guohui Li et al.), which describes either sterilization in wet state using a cobalt radiation source, with subsequent cold-temperature storage, or freeze-drying with subsequent storage in glycerine at room temperature.
Conservation using glycerine is also recommended for the human placenta (amnion) used for allotransplants by the Deutches Institut fur Zell- and Gewebeersatz gGmbH (Delitzcher St 141, 04129 Leipzig, SRN).
Document UA 12391U (E. Y. Fistal et al.) describes the healing of necrotic wounds after deep burns using freeze-dried pig skin.
Biological wound dressings based on collagen for the healing of wounds, including burns, are also described in documents RU 2185179 and RU 2124354.
The problem of sterility and shelf life can be attenuated by the removal of cells from the transplant, which will thus become partially or entirely acellular. One effort at solving this problem can be found in the patent document No. CN 20031124306 (Hu Jie), describing xenotransplant as a biological dressing for wounds and burns. Animal tissue, such as skin, small intestine wall or placenta, can be partially rid of cells according to the above invention by using water and detergent solution, and this is done on the surface which will be in contact with the wound. The cellular structure of other parts, such as the epidermis, will remain preserved. Then the tissue will be crosslinked using an appropriate agent, such as glutaraldehyde, will be washed out, and will be stored in wet state at a temperature below 4° C.
Another document, CN 20051126108 (Dong Qun Lin), describes the manner of cell removal from mammalian skin through the repeated action of 2N to 5N NaOH solution, followed by washing in a detergent solution and in water.
Another document, CN20041022506 (Dai Weihua et al.), describes the manner of preparation of a biodegradable acellular dermis using the combined action of enzymes, alkalis, and other chemical agents.
Published application US 20050186286 (Yoshihiro Takami) describes the method of cell removal from mammalian (eg. human or pig) skin using a combined action of proteolytic enzymes and detergents, whereas thus prepared skin is designated for use as an allotransplant or xenotransplant for burns healing. Sterilization is done by a subsequent immersion of the acellular dermis into an azide solution.
Similar acellular xenodermic matrix is OASIS, made by AelsLife, which provides a framework for a three-dimensional migration of cells. This biological wound cover, which according to the manufacturer contains important non-cellular compounds and structures present in live skin, is made by lyophylization of porcine dermis after the cells are removed using enzymes and detergents.
Biosynthetic bandage E*Z DERM, by manufacturer Brennen Medical Inc., uses a porcine dermis xenograft, treated by crosslinking of collagen using aldehydes.
Patent document JP19900247300 (Koide Mikio) describes a biological cover using a denatured collagen matrix, formed from acellular bovine dermis through crosslinking and heat-induced denaturation of collagen structures. This structure is, according to the cited invention, appropriate for seeding using autologous keratinocytes for higher healing efficacy.
Other efforts to resolve the problem were various semi-synthetic skin substitutes, for example a scaffold from a reconstituted bovine collagen, seeded with human fibroblasts (i.e. the above-mentioned INTEGRA dressing.)
Another example of a combined transplant is the “recombined skin: (RK) according to the CZ Patent No. 281176. RK is prepared using cultivation of human keratinocytes on a cell-free porcine-dermis. Burns 1993; 19: 118-23). The dried dermis is used for the cultivation of human keratinocytes and after cultivation the dermis, with a keratinocyte layer (or RK), is detached from the Petri dish and applied to the wound. RK is applied with the keratinocytes in contact with the wound, and the dermis on the outside (“upside down”). As compared to simple epidermal grafts, the RK shows an advantage of higher durability, the detachment from the Petri dish without enzymatic action, and easy manipulation. An advantage as compared to keratinocyte cultures on synthetic substrates and collagen-based gels is that RK's consistency is similar to skin, and this results in excellent wound adhesion and a hemostatic effect. It's possible to make RK using both autologous and allogenic keratinocytes. Keratinocytes are cultured on the epidermal side of epidermis, i.e. where the basal membrane divides the dermis from the epidermis. The inventor of the above cited invention mentions that the cell-free dermis can be sterilized using gamma radiation for better shelf-life at room temperature and higher safety. A disadvantage of thus gamma-sterilized dermis is, however, its partial degradation and loss of durability in wet state.
A similar combined biological burn dressing is described in the patent application TW20000118374 (Yang Mei-Ru et al.), where live human fibroblasts in an acellular porcine dermis are combined with human keratinocytes cultured on the basal-membrane side of an acellular matrix.
A general problem, which prevents a significant acceptance of use of these biological materials, is the fact that it is impossible to use a routine and reliable sterilization process. Another specific problem which prevents wider use of the above-mentioned materials is their limited or demanding shelf-life, and last but not least, their manufacturing cost. A difficult problem is also displayed by dehydrated materials which swell isotropically during rehydration, i.e. the (relatively) equal increase of all of the transplant dimensions after rehydration. The present invention brings a solution to these problems.